PROJECT SUMMARY: Glioblastomas (GBMs) are aggressive brain tumors with poor response to conventional treatments. Novel therapies that stimulate immune responses are showing great promise to treat other solid tumors but have limited effect against GBM. This is due, in large part, to the presence of a sizable population of tumor-associated microglia/macrophages (TAMs) that are induced by the tumor cells to acquire an immunosuppressive, tumor- promoting phenotype. The suppression of innate and adaptive immunity caused by TAMs, coupled with the substantial intra-tumoral heterogeneity of GBM, facilitates the escape of malignant cells that cause unavoidable recurrence. Therefore, there is a dire need for novel targeted approaches that can create a persistent anti-tumor immune environment and at the same time overcome GBM heterogeneity. The extracellular matrix (ECM) is a unique target accessible outside the cells, widespread in the tumor, and less heterogeneous than the tumor cells that it surrounds. Accordingly, we propose a novel anti-tumor approach based on ECM targeting to reduce immunosuppression. Specifically, we will investigate the immunomodulatory functions and targeting value of the ECM protein fibulin-3, which is a major tumor-promoting factor secreted by GBM cells but absent from normal brain. Preliminary evidence suggests a positive correlation between fibulin-3 and immunosuppressive signals in GBM, as well as signs of immune activation following fibulin-3 disruption. Accordingly, we hypothesize that fibulin-3 promotes TAM immunosuppression, contributing to GBM immune escape and progression. To validate this hypothesis, our first Specific Aim is to investigate the effect of fibulin-3 downregulation on TAM polarization. We will first disrupt fibulin-3 signaling in GBM cells to analyze its regulatory effect on immunosuppressive cytokines and immune checkpoint ligands. Next, we will use a model for inducible downregulation of fibulin-3 in vitro and in vivo, to test if the loss of this protein enhances an inflammatory TAM phenotype, causing tumor-lytic effects. In our second Specific Aim, we will investigate whether fibulin-3 blockade enhances anti-tumor immunity. We have developed a function-blocking anti-fibulin-3 antibody that reduces the growth of GBM and, surprisingly, increases TAM infiltration and tumor necrosis. Using GBM / TAM co-cultures and intracranial xenograft models, we will investigate if anti-fibulin-3 can reduce immunosuppressive signals from GBM cells and rescue the undesirable tumor-promoting TAM phenotype into a desired anti-tumor phenotype, without negative systemic effects. Finally, using syngeneic GBMs models, we will test if anti-fibulin-3 can also increase the intratumoral infiltration of activated T cells for a sustained anti-tumor response. Successful completion of this project will validate fibulin-3 as a novel immunomodulatory factor in the microenvironment of GBM, which can be targeted to overcome the heterogeneity of GBM cells and potentiate anti-tumor immunity.